Project Summary/Abstract Studying normal epigenomic variation among human populations, and the proximate and evolutionary origins of that variation, is critical to understanding the effects of epigenomic processes on both human disease and normal phenotypes. Epigenetic marks like methylation are dynamic and can be influenced by the environment, providing relatively rapid adjustment to variation in environmental conditions. Therefore, studying how these marks change in response to stimuli leads us to understanding physiological and metabolic responses to major variation in environmental factors and/or lifestyles throughout human evolution. Differences in subsistence strategy, e.g. hunting and gathering versus agriculture, results in differences in most aspects of human existence like living environment, activity level, and nutritional intake. For the Batwa rainforest hunter-gatherers and the Bakiga agriculturalists from Uganda, these differences have led to major phenotypic variation in body size ? the Batwa are substantially shorter than the Bakiga. Their height difference is due, in part, to a heritable genomic component, and also potentially to documented epigenetic differences near genes related to growth and development. This proposal describes a complementary addition to an established and fully-funded project, in which the cells of 50-89 Batwa and Bakiga individuals will be challenged with growth hormones, followed by RNAseq to identify genome-wide population differences in gene expression. The proposed complementary project will use these same cells to characterize baseline and dynamic growth hormone response differences in DNA methylation between the Batwa and the Bakiga that may mediate the gene expression and phenotypic differences between populations. First, I will identify differentially methylated sites (DMS) and genes i) at baseline between the Batwa and the Bakiga, ii) across treated and untreated cells that are population dependent, and lastly, iii) between baseline and treatments irrespective of population. Second, when the cells are stimulated with growth hormone, genes identified for changes in methylation will be correlated with changes in gene expression. Additionally, I will identify over- and under-represented biological pathways among the DMS, and genes with significant correlations of methylation status-gene expression. Lastly, I will use previously collected Batwa and Bakiga anthropometric and 1M SNP genotype data to i) identify methylation QTLs (meQTLs) for DMS, ii) characterize the evolutionary histories of the meQTLs relative to genome-wide patterns, at baseline between the populations and across the response to GH stimulation, and iii) compare the DMS identified from the growth hormone stimulation to the previously identified genomic regions associated with the Batwa pygmy phenotype. These unique samples and integrated analyses represent a major opportunity for a functional and evolutionary genomics investigation of body size. This project will provide important, novel insights into the static and dynamic regulation of the major genomic pathways moderating human growth and development in response to differing environments and lifestyles.